The genus Potyvirus, which is the largest group of plant viruses, has a broad host range. Potato virus Y (hereinafter referred to as PVY), which is a virus that belongs to the genus Potyvirus, can non-persistently transmit through aphids and infect a variety of solanaceous plant species. When it comes to tobacco, PVY causes symptoms such as reduced plant height and vein necrosis. This results in decreases in quality and yield of leaf tobacco and thus causes great damage to tobacco production in the world. From leaf tobacco infected with PVY and decreased in quality, tobacco products with significantly decreased quality are produced.
Meanwhile, Tobacco bushy top virus (hereinafter referred to as TBTV), which is a virus that belongs to the genus Umbravirus, is known as a causative virus for tobacco bushy top disease that has occurred in Africa and Asia. TBTV is persistently transmitted by aphids at a natural environment and causes plant stunting and leaf mottling symptom of tobacco. This results in decreases in quality and yield of tobacco. In particular, the tobacco bushy top disease has become an important disease in African countries.
In tobacco (Nicotiana tabacum), Virgin A mutant (hereinafter referred to as VAM), which is a genetic resource exhibiting resistance to PVY, is known and has been frequently utilized in a tobacco breeding program. However, the VAM-Breaking strain (PVY-Breaking strain which can also be expressed as PVY-B), which is a new strain of PVY to break the resistance of VAM, has recently been reported around the world. Currently, there has been strong demand for tobacco resistant to the VAM-Breaking strain. Recently, tobacco acquiring resistance to the VAM-Breaking strain by gamma-ray irradiation has been reported (Non-Patent Literature 1), but a causative gene has not been identified. Further, some of wild species including, for example, Nicotiana africana have been known to exhibit resistance to the PVY-Breaking strain, but have not yet been utilized in a breeding program.
Still further, it has been reported that exploration for resistance sources to tobacco bushy top disease was conducted using 43 types of tobacco varieties and wild species belonging to the genus Nicotiana, and none of these tobacco varieties exhibited resistance to tobacco bushy top disease, while several wild species did not show any symptoms of viral diseases (Non-Patent Literature 2). However, the mode of inheritance of resistance of such wild species has not been elucidated, and it is expected that introduction of the resistant trait into the cultivated varieties of N. tabacum from the wild species is accompanied by introduction of a trait that adversely affects quality and yield. Therefore, a practical use of them is a long way off.
About half of approximately 200 types of known plant virus-resistance genes are recessively inherited (Non-Patent Literature3). These genes are considered to be host factors required for, for example, replication and cell-to-cell movement of viruses. Research over the past decade has revealed some of these factors. For example, translation initiation factors such as eIF4E and eIF4G, DEAD-box RNA helicase-like protein (Non-Patent Literature 4), a cysteine-rich VPg-interacting protein (Non-Patent Literature 5), Translation elongation factor (Non-Patent Literature 6), and others have been identified as recessive virus-resistance gene factors. As a matter of course, these factors are not all of the virus-resistance genes. In addition, a number of other factors are considered to be candidates for the virus-resistance genes (Non-Patent Literature 3). Examples of such candidates include various plant factors associated with sieve tube transport of plant viruses (Non-Patent Literature 7).
Viruses utilize translation initiation systems of hosts in the synthesis of viral proteins. In 2002, it was shown that a recessive resistance gene factor against Turnip mosaic virus (TuMV) in Arabidopsis thaliana is a mutation of a translation initiation factor eIF(iso)4E (Non-Patent Literature 8). Since then, the association of the eIF4E gene family with recessive resistance to known viruses belonging to the genus Potyvirus has been studied in some plants. In fact, it has been shown that recessive virus-resistance is acquired by an artificial mutation of eIF4E or eIF(iso)4E.
For example, Patent Literature 1 describes a method of imparting virus resistance by suppressing the eIF4E gene (not including eIF(iso)4E) function. Further, Patent Literature 2 describes a mutant plant having eIF4E or eIF(iso)4E not be acted upon by a virus, by splicing mutation of the eIF4E gene or eIF(iso)4E gene. The mutation is insertion, deletion, or substitution of at least one base in a non-coding region of eIF4E or eIF(iso)4E or in a splicing element (a region containing ±10 bases from a boundary site between an exon and an intron) of eIF4E or eIF(iso)4E, and the mutation is intended to occur preferably in an intron, and more preferably in a first intron. Still further, Patent Literature 3 describes a method involving selection of pepper veinal mottle disease (Pepper veinal mottle virus (PVMV)) resistant plant by combination of mutations both in eIF4E and in eIF(iso)4E, and specifically describes a method of selecting a plant in which eIF4E and eIF(iso)4E are not expressed at all, but mutated eIF4E does express.
Yet further, it has been shown that, for example, a causative gene in recessive resistance to PVY in pepper is eIF4E (Non-Patent Literature 9). In addition, it has been shown that Clover yellow vein virus proliferates in eIF(iso)4E-deficient Arabidopsis thaliana, but does not proliferate in eIF4E-deficient Arabidopsis thaliana, and, on the contrary, TuMV proliferates in the eIF4E-deficient Arabidopsis thaliana, but does not proliferate in the eIF(iso)4E-deficient Arabidopsis thaliana (Non-Patent Literature 10). Further, in order to acquire resistance to PVMV, both eIF4E and eIF(iso)4E must lose their functions simultaneously (Non-Patent Literature 11). For example, Non-Patent Literature 12, Non-Patent Literature 13, and others have reviewed recent translation initiation factors and plant viral resistance.
Association between a limited number of viruses other than the viruses belonging to the genus Potyvirus and translation initiation factors have also been pointed out. For example, Cucumber mosaic virus (CMV) is a virus belonging to the genus Cucumovirus. Production of 3a protein that is associated with cell-to-cell movement of CMV is inhibited in Arabidopsis thaliana in which eIF4E or eIF4G is destroyed. In addition, Rice yellow mottle virus (RYMV) is a virus belonging to the genus Sobemovirus. Rice in which eIF(iso)4G has a mutation is resistant to RYMV (Non-Patent Literature 14 and Non-Patent Literature 15).
As for a tomato, which is a solanaceous plant as is the case with tobacco, a relationship between Potyvirus resistance and a translation initiation factor eIF4E has been studied based on an exhaustive analysis of a tomato mutant panel. In the study, it has been shown that suppression of the function of eIF4E1, which is a member of the eIF4E gene family, imparts resistance to PVY and Pepper mottle virus (PepMoV), but does not impart resistance to Tobacco etch virus (TEV) (Non-Patent Literature 16). It has also been shown together that suppression of the function of eIF4E2, eIF(iso)4E, eIF4G, or eIF(iso)4G do not impart resistance to these viruses belonging to the genus Potyvirus. It has also been shown that simultaneous suppression of the functions of eIF4E1 and eIF4E2 by using RNAi (RNA interference) imparts resistance to seven types of viruses belonging to the genus Potyvirus, including PVY, PepMoV and TEV. However, interestingly, it has been shown that suppression of the function of eIF(iso)4E by RNAi does not impart resistance to any of these viruses (Non-Patent Literature 17). It has also been shown together that eIF(iso)4E of tomato is not associated with resistance to viruses other than the viruses belonging to the genus Potyvirus (Non-Patent Literature 17).
Thus, an association between PVY resistance and eIF4E in any plant has been pointed out so far, but an association between PVY resistance and eIF(iso)4E has never been reported. Although eIF(iso)4E is categorized into the eIF4E family, DNA sequence identity between eIF4E and eIF(iso)4E in plants is generally less than 60%. In addition, eIF(iso)4E forms a translation complex different from that formed by eIF4E. Specifically, eIF(iso)4E, together with eIF(iso)4G, forms a translation complex eIF(iso)4F, while eIF4E, together with eIF4G, forms a translation complex eIF4F.
As for tobacco (Nicotiana tabacum), reduction of an expression level of eIF4E1 or eIF(iso)4E has been reported (Non-Patent Literature 18). In this report, the transcription of eIF4E1 or eIF(iso)4E of tobacco is suppressed by using antisense technology. Non-Patent Literature 18 describes that production of tobacco in which the amount of transcripts of eIF4E1 is suppressed to 30% to 40% relative to a control and tobacco in which the amount of transcripts of eIF(iso)4E is suppressed to 60% relative to a control and that in a crossbreed progeny between both of the above tobaccos, the amount of transcripts of eIF4E1 is reduced to 26% relative to a control, and the amount of transcripts of eIF(iso)4E is reduced to 31% relative to a control. However, Non-Patent Literature 18 does not describe association with viral resistance at all. Further, the possibility that HC-Pro protein of PVY interacts with eIF(iso)4E of tobacco has been suggested from an assay system using Nicotiana benthamiana (Non-Patent Literature 19). However, Non-Patent Literature 19 has not pointed out association with resistance.
Further, as for tobacco, it has been found from a comprehensive analysis of transcripts of PVY-resistant VAM tobacco and transcripts of PVY-sensitive tobacco that the level of transcripts of eIF4E gene is specifically low in VAM tobacco, and indeed, it has been shown that tobacco having a mutation in this gene becomes PVY resistance (Non-Patent Literature 20).
On the contrary, it has been reported that there is no association between Potyvirus resistance of tobacco and mutations in the translation initiation factors eIF4E and eIF(iso)4E (Non-Patent Literature 21). This report has investigated base sequences of eIF4E and eIF(iso)4E genes in varieties resistant to PVY and PepMoV, which are viruses belonging to the genus Potyvirus and can infect tobacco, and in varieties sensitive to these viruses. As a result, it has been shown that an association between mutations that occurred in both of the genes and resistance and sensitivity to the viruses was not observed. For example, while no mutations were detected in the eIF4E gene and eIF(iso)4E gene of a certain PVY-resistant variety, mutations were observed in those of a PVY-sensitive variety. In similar experiments conducted on other plants in the past, mutations were detected in eIF4E and eIF(iso)4E genes of a PVY-resistant variety, and the PVY resistance was attributed to such mutations. Thus, Non-Patent Literature 21 concludes that, in tobacco, unlike other solanaceous plants, there is no association between the PVY resistance and the translation initiation factors eIF4E and eIF(iso)4E. As discussed above, as for tobacco, unlike other plants, the issue of an association between Potyvirus resistance and the translation initiation factors is still in chaos.
Furthermore, an association between the translation initiation factors and resistance to viruses belonging to the genus Umbravirus is not known at all in any plant species including tobacco.
Tobacco (Nicotiana tabacum), which is an amphidiploid, has a larger number of genes than a normal diploid plant. In tobacco, basically one pair of genes derived from Nicotiana sylvestris and one pair of genes derived from Nicotiana tomentosiformis are present. That is, in tobacco, at least two pairs of homologous genes are present. Therefore, the mode of inheritance of tobacco is more complicated than that of diploid plants. In Arabidopsis thaliana, three types of eIF4E and one type of eIF(iso)4E are supposed to be present (Non-Patent Literature 12). In tobacco, translation initiation factors, which are observed in tobacco, are considered to be all present in pairs. It has been found that an eIF4E family of tobacco has at least 12 members including even a cap-binding protein functionally similar to eIF4E (Non-Patent Literature 20). The members of the gene family of tobacco translation initiation factors are much larger in number when further including even eIF4G and eIF(iso)4G.